Angiogenesis is the process of new blood vessel formation by degradation of extracellular matrix (ECM), migration, division, and differentiation by pre-existing vascular endothelial cells. Angiogenesis is involved in various physiological and pathological events, such as embryonic development, wound healing, tumor growth, chronic inflammation, obesity, etc. Angiogenesis includes the proliferation of vascular endothelial cells and their migration from the blood vessel wall to the surrounding tissue following the source of the angionenic stimuli. Sequentially, the activation of various proteases helps the vascular endothelial cells to degrade the basement membrane and form loops. These formed loops differentiate into new vessels.
The angiogenic process is known to be strictly regulated by various types of angiogenic simulators and inhibitors. Angiogenesis does not occur in a normal state due to a quantitative balance between angiogenic inhibitors, such as thrombospondin-1, platelet factor-4, angiostatin, etc., and angiogenic stimulators, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), etc. However, when a wound or tumor occurs, for the wound healing or tumor growth, the above balance is upset to enable new blood vessels to grow. The formation involves an overexpression of angiogenic stimulators.
Angiogenesis is an essential step for tissue regeneration, as well as wound healing. For example, a placenta in which angiogenesis is underdeveloped is an important cause of miscarriage. Necrosis, ulcer, and ischemia caused by non-formation of vessels cause malfunction of tissues or organisms, or can lead to death. In addition, atherosclerosis, myocardial infarction, and angina pectoris are due to an inadequate blood supply. Accordingly, treatment methods of reducing tissue damage caused by hypoxia or undernutrition due to incomplete blood vessel formation, while inducing or stimulating neovascularization for proper tissue regeneration, are needed.
A therapy of treating diseases using angiogenesis is called an angiogenic therapy. VEGF, an angiogenic simulator, is used as a therapeutic agent for severe local anemia. In addition, angiogenic simulators, such as FGF, epidermal growth factor (EGF) and platelet-derived endothelial growth factor (PDEGF), are also being studied for clinical treatment. However, the above factors are disadvantageous for clinical applications because they are proteins which are difficult and costly to isolate and purify.
In 1997, Asahara and colleagues reported that a purified population of CD34+ hematopoietic progenitor cells isolated from the circulation system of adults could be in vitro differentiated into endothelial lineage cells named endothelial progenitor cells (EPCs). Based on the above, bone marrow-derived cells and EPCs proliferated ex vivo were used in the treatment of limb ischemia and the regeneration of heart muscles The EPCs were tried in auto-transplantation for blood vessel regeneration. After that, it was reported that not only stromal vascular fraction (SVF) in the adipose tissue but mesenchymal stem cells (MSCs) found in bone marrow and umbilical cord blood could also be differentiated into vascular endothelial cells. Adipose stem cells could be differentiated ex vivo into vascular endothelial cells and showed early angiogenesis activity in ischemia animal models.
However, because stem cells are individually transplanted in animal models of ischemia using MSCs, most reports so far have said that growth factors secreted from the stem cells, rather than the stem cells themselves, induce angiogenesis of the host. Some stem cells are introduced into the newly formed blood vessels but there have been no reports that stem cells per se induce angiogenesis. There has also been a report that when cells produced by decomposing adipose tissues were transplanted into animals without culturing the stromal vascular fraction (SVF) therefrom, it was possible to differentiate them into vascular endothelial cells. However, since the above method did not induce proliferation of adipose stem cells via subculturing, the amount of vascular endothelial cells differentiated from the adipose stem cells was very small. In particular, since the differentiated vascular endothelial cell showed low levels of proliferation and differentiation, the application is limited.
Therefore, the present inventors conducted extensive research on an angiogenic therapy using stem cells for effectively inducing angiogenesis of stem cells transplanted in the body. As a result, the present inventors found that, if stem cells are cultured on a culture plate with a surface of a hydrophobic property by physically attaching the cells to the culture plate via cell-matrix interactions, or they are cultured where they are bonded to growth factors immobilized to the surface of the culture plate via their interaction with the growth factors, stem cells proliferate while being attached to the surface of the culture plate initially, while the proliferated stem cells are later detached from the surface of the culture plate to form a three-dimensional cell cluster as the intercellular interaction becomes stronger than the cell-matrix interaction under high cellular density. The present inventors further discovered that the stem cells within the thus formed cell cluster not only secrete angiogenic stimulators, but are also differentiated into vascular cells. Based on the above findings, the present inventors developed a method of using a cell cluster composed of vascular cells differentiated from stem cells as a cell therapy agent for angiogenesis to achieve the present invention.